EP1526186A2 - Pronostic de cancer colorectal - Google Patents

Pronostic de cancer colorectal Download PDF

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EP1526186A2
EP1526186A2 EP04251933A EP04251933A EP1526186A2 EP 1526186 A2 EP1526186 A2 EP 1526186A2 EP 04251933 A EP04251933 A EP 04251933A EP 04251933 A EP04251933 A EP 04251933A EP 1526186 A2 EP1526186 A2 EP 1526186A2
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seq
genes
portfolio
gene
combination
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EP1526186A3 (fr
EP1526186B1 (fr
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Yixin Wang
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Janssen Diagnostics LLC
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
    • A01M1/00—Stationary means for catching or killing insects
    • A01M1/08—Attracting and catching insects by using combined illumination or colours and suction effects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

Definitions

  • This invention relates to prognostics for colorectal cancer based on the gene expression profiles of biological samples.
  • Colorectal cancer is a heterogenous disease with complex origins. Once a patient is treated for colorectal cancer, the likelihood of a recurrence is related to the degree of tumor penetration through the bowel wall and the presence or absence of nodal involvement. These characteristics are the basis for the current staging system defined by Duke's classification.
  • Duke's A disease is confined to submucosa layers of colon or rectum.
  • Duke's B tumor invades through muscularislitis and could penetrate the wall of colon or rectum.
  • Duke's C disease includes any degree of bowel wall invasion with regional lymph node metastasis.
  • Surgical resection is highly effective for early stage colorectal cancers, providing cure rates of 95% in Duke's A and 75% in Duke's B patients.
  • the presence of positive lymph node in Duke's C disease predicts a 60% likelihood of recurrence within five years.
  • Treatment of Duke's C patients with a postsurgical course of chemotherapy reduces the recurrence rate to 40%-50%, and is now the standard of care for Duke's C patients.
  • the benefit of postsurgical chemotherapy in Duke' B has been harder to detect and remains controversial.
  • the Duke's B classification is imperfect as approximately 20 - 30% of these patients behave more like Duke's C and relapse within a 5 year timeframe. There is clearly a need to identify better prognostic factors than nodal involvement for guiding selection of Duke's B into those that are likely to relapse and those that will survive.
  • the invention is a method of assessing the likelihood of a recurrence of colorectal cancer in a patient diagnosed with or treated for colorectal cancer.
  • the method involves the analysis of a gene expression profile.
  • the gene expression profile includes at least three genes.
  • the gene expression profile includes at least four genes.
  • Articles used in practicing the methods are also an aspect of the invention.
  • Such articles include gene expression profiles or representations of them that are fixed in machine-readable media such as computer readable media.
  • Articles used to identify gene expression profiles can also include substrates or surfaces, such as microarrays, to capture and/or indicate the presence, absence, or degree of gene expression.
  • kits include reagents for conducting the gene expression analysis prognostic of colorectal caner recurrence.
  • nucleic acid sequences having the potential to express proteins, peptides, or mRNA such sequences referred to as "genes"
  • genes such sequences referred to as "genes”
  • assaying gene expression can provide useful information about the occurrence of important events such as tumerogenesis, metastasis, apoptosis, and other clinically relevant phenomena. Relative indications of the degree to which genes are active or inactive can be found in gene expression profiles.
  • the gene expression profiles of this invention are used to provide a prognosis and treat patients for colorectal cancer.
  • Sample preparation requires the collection of patient samples.
  • Patient samples used in the inventive method are those that are suspected of containing diseased cells such as epithelial cells taken from a colon sample or from surgical margins.
  • One useful technique for obtaining suspect samples is Laser Capture Microdisection (LCM).
  • LCM technology provides a way to select the cells to be studied, minimizing variability caused by cell type heterogeneity. Consequently, moderate or small changes in gene expression between normal and cancerous cells can be readily detected.
  • the samples comprise circulating epithelial cells extracted from peripheral blood. These can be obtained according to a number of methods but the most preferred method is the magnetic separation technique described in U.S. Patent 6,136,182 assigned to Immunivest Corp which is incorporated herein by reference.
  • Preferred methods for establishing gene expression profiles include determining the amount of RNA that is produced by a gene that can code for a protein or peptide. This is accomplished by reverse transcriptase PCR (RT-PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify complimentary DNA (cDNA) or complimentary RNA (cRNA) produced from mRNA and analyze it via microarray. A number of different array configurations and methods for their production are known to those of skill in the art and are described in U.S.
  • Patents such as: 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637; the disclosures of which are incorporated herein by reference.
  • Microarray technology allows for the measurement of the steady-state mRNA level of thousands of genes simultaneously thereby presenting a powerful tool for identifying effects such as the onset, arrest, or modulation of uncontrolled cell proliferation.
  • Two microarray technologies are currently in wide use. The first are cDNA arrays and the second are oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same. The product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to nucleic acid sequence at a known location on the microarray.
  • the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA, expressed in the sample cells.
  • mRNA mRNA
  • Analysis of the expression levels is conducted by comparing such intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a diseased tissue can be compared with the expression intensities generated from normal tissue of the same type (e.g., diseased colon tissue sample vs. normal colon tissue sample). A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.
  • Gene expression profiles can also be displayed in a number of ways.
  • the most common method is to arrange a raw fluorescence intensities or ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes.
  • the data is arranged so genes that have similar expression profiles are proximal to each other.
  • the expression ratio for each gene is visualized as a color. For example, a ratio less than one (indicating down-regulation) may appear in the blue portion of the spectrum while a ratio greater than one (indicating up-regulation) may appear as a color in the red portion of the spectrum.
  • Commercially available computer software programs are to display such data including "GENESPRINT” from Silicon Genetics, Inc. and “DISCOVERY” and "INFER” software from Partek, Inc..
  • the genes that are differentially expressed are either up regulated or down regulated in patients with a relapse of colon cancer relative to those with a relapse.
  • Up regulation and down regulation are relative terms meaning that a detectable difference (beyond the contribution of noise in the system used to measure it) is found in the amount of expression of the genes relative to some baseline.
  • the baseline is the measured gene expression of a non-relapsing patient.
  • the genes of interest in the diseased cells are then either up regulated or down regulated relative to the baseline level using the same measurement method.
  • Diseased in this context, refers to an alteration of the state of a body that interrupts or disturbs, or has the potential to disturb, proper performance of bodily functions as occurs with the uncontrolled proliferation of cells.
  • someone is diagnosed with a disease when some aspect of that person's genotype or phenotype is consistent with the presence of the disease.
  • the act of conducting a diagnosis or prognosis includes the determination of disease/status issues such as determining the likelihood of relapse and therapy monitoring.
  • therapy monitoring clinical judgments are made regarding the effect of a given course of therapy by comparing the expression of genes over time to determine whether the gene expression profiles have changed or are changing to patterns more consistent with normal tissue.
  • levels of up and down regulation are distinguished based on fold changes of the intensity measurements of hybridized microarray probes.
  • a 2.0 fold difference is preferred for making such distinctions or a p-value less than .05. That is, before a gene is said to be differentially expressed in diseased/relapsing versus normal/non-relapsing cells, the diseased cell is found to yield at least 2 more, or 2 times less intensity than the normal cells. The greater the fold difference, the more preferred is use of the gene as a diagnostic or prognostic tool.
  • Genes selected for the gene expression profiles of the instant invention have expression levels that result in the generation of a signal that is distinguishable from those of the normal or non-modulated genes by an amount that exceeds background using clinical laboratory instrumentation.
  • Statistical values can be used to confidently distinguish modulated from non-modulated genes and noise. Statistical tests find the genes most significantly different between diverse groups of samples. The Student's t-test is an example of a robust statistical test that can be used to find significant differences between two groups. The lower the p-value, the more compelling the evidence that the gene is showing a difference between the different groups. Nevertheless, since microarrays measure more than one gene at a time, tens of thousands of statistical tests may be asked at one time. Because of this, one is unlikely to see small p-values just by chance and adjustments for this using a Sidak correction as well as a randomization/permutation experiment can be made.
  • a p-value less than .05 by the t-test is evidence that the gene is significantly different. More compelling evidence is a p-value less then .05 after the Sidak correction is factored in. For a large number of samples in each group, a p-value less than 0.05 after the randomization/permutation test is the most compelling evidence of a significant difference.
  • Another parameter that can be used to select genes that generate a signal that is greater than that of the non-modulated gene or noise is the use of a measurement of absolute signal difference.
  • the signal generated by the modulated gene expression is at least 20% different than those of the normal or non-modulated gene (on an absolute basis). It is even more preferred that such genes produce expression patterns that are at least 30% different than those of normal or non-modulated genes.
  • Genes can be grouped so that information obtained about the set of genes in the group provides a sound basis for making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice. These sets of genes make up the portfolios of the invention. In this case, the judgments supported by the portfolios involve colorectal cancer. As with most diagnostic markers, it is often desirable to use the fewest number of markers sufficient to make a correct medical judgment. This prevents a delay in treatment pending further analysis as well inappropriate use of time and resources.
  • portfolios are established such that the combination of genes in the portfolio exhibit improved sensitivity and specificity relative to individual genes or randomly selected combinations of genes.
  • the sensitivity of the portfolio can be reflected in the fold differences exhibited by a gene's expression in the diseased state relative to the normal state.
  • Specificity can be reflected in statistical measurements of the correlation of the signaling of gene expression with the condition of interest. For example, standard deviation can be a used as such a measurement. In considering a group of genes for inclusion in a portfolio, a small standard deviation in expression measurements correlates with greater specificity. Other measurements of variation such as correlation coefficients can also be used in this capacity.
  • a preferred method of establishing gene expression portfolios is through the use of optimization algorithms such as the mean variance algorithm widely used in establishing stock portfolios. This method is described in detail in the patent application entitled “Portfolio Selection” by Tim Jatkoe, et, al., filed on March 21,2003. Essentially, the method calls for the establishment of a set of inputs (stocks in financial applications expression as measured by intensity here) that will optimize the return (e.g., signal that is generated) one receives for using it while minimizing the variability of the return. Many commercial software programs are available to conduct such operations. "Wagner Associates Mean-Variance Optimization Application”, referred to as “Wagner Software” throughout this specification, is preferred. This software uses functions from the “Wagner Associates Mean-Variance Optimization Library” to determine an efficient frontier and optimal portfolios in the Markowitz sense is preferred.
  • microarray data be transformed so that it can be treated as an input in the way stock return and risk measurements are used when the software is used for its intended financial analysis purposes.
  • Wagner Software is employed in conjunction with microarray intensity measurements the following data transformation method is employed.
  • Genes are first pre-selected by identifying those genes whose expression shows at least some minimal level of differentiation.
  • the preferred pre-selection process is conducted as follows.
  • a baseline class is selected. Typically, this will comprise genes from a population that does not have the condition of interest. For example, if one were interested in selecting a portfolio of genes that are diagnostic for relapsing colon cancer, samples from patients without relapses can be used to make the baseline class.
  • the baseline class is selected, the arithmetic mean and standard deviation is calculated for the indicator of gene expression of each gene for baseline class samples. This indicator is typically the fluorescent intensity of a microarray reading.
  • the statistical data computed is then used to calculate a baseline value of (X*Standard Deviation + Mean) for each gene.
  • X is a stringency variable selected by the person formulating the portofolio. Higher values of X are more stringent than lower. Preferably, X is in the range of .5 to 3 with 2 to 3 being more preferred and 3 being most preferred.
  • Ratios between each experimental sample (those displaying the condition of interest) versus baseline readings are then calculated.
  • the ratios are then transformed to base 10 logarithmic values for ease of data handling by the software. This enables down regulated genes to display negative values necessary for optimization according to the Markman mean-variance algorithm using the Wagner Software.
  • the preprocessed data comprising these transformed ratios are used as inputs in place of the asset return values that are normally used in the Wagner Software when it is used for financial analysis purposes.
  • an optimized portfolio is selected for a given input level (return) or variance that corresponds to a point on the frontier.
  • inputs or variances are the predetermined standards set by the person formulating the portfolio.
  • one seeking the optimum portfolio determines an acceptable input level (indicative of sensitivity) or a given level of variance (indicative of specificity) and selects the genes that lie along the efficient frontier that correspond to that input level or variance.
  • the Wagner Software can select such genes when an input level or variance is selected. It can also assign a weight to each gene in the portfolio as it would for a stock in a stock portfolio.
  • Determining whether a sample has the condition for which the portfolio is diagnostic can be conducted by comparing the expression of the genes in the portfolio for the patient sample with calculated values of differentially expressed genes used to establish the portfolio.
  • a portfolio value is first generated by summing the multiples of the intensity value of each gene in the portfolio by the weight assigned to that gene in the portfolio selection process.
  • a boundary value is then calculated by (Y* standard deviation + mean of the portfolio value for baseline groups) where Y is a stringency value having the same meaning as X described above.
  • a sample having a portfolio value greater than the portfolio value of the baseline class is then classified as having the condition. If desired, this process can be conducted iteratively in accordance with well known statistical methods for improving confidence levels. Optionally one can reiterate this process until best prediction accuracy is obtained.
  • the process of portfolio selection and characterization of an unknown is summarized as follows:
  • genes can first be pre-selected by identifying those genes whose expression shows some minimal level of differentiation.
  • the pre-selection in this alternative method is preferably based on a threshold given by where ⁇ t is the mean of the subset known to possess the disease or condition, ⁇ n is the mean of the subset of normal samples, and ⁇ t + ⁇ n represent the combined standard deviations.
  • a signal to noise cutoff can also be used by pre-selecting the data according to a relationship such as This ensures that genes that are pre-selected based on their differential modulation are differentiated in a clinically significant way. That is, above the noise level of instrumentation appropriate to the task of measuring the diagnostic parameters.
  • a matrix is established in which columns represents samples, rows represent markers and each element is a normalized intensity measurement for the expression of that marker according to the relationship: where I is the intensity measurement.
  • portfolio size can be limited to a fixed range or number of markers. This can be done either by making data pre-selection criteria more stringent (e.g, instead of or by using programming features such as restricting portfolio size.
  • the process of selecting a portfolio can also include the application of heuristic rules.
  • such rules are formulated based on biology and an understanding of the technology used to produce clinical results. More preferably, they are applied to output from the optimization method.
  • the mean variance method of portfolio selection can be applied to microarray data for a number of genes differentially expressed in subjects with colorectal cancer. Output from the method would be an optimized set of genes that could include some genes that are expressed in peripheral blood as well as in diseased tissue. If samples used in the testing method are obtained from peripheral blood and certain genes differentially expressed in instances of breast cancer could also be differentially expressed in peripheral blood, then a heuristic rule can be applied in which a portfolio is selected from the efficient frontier excluding those that are differentially expressed in peripheral blood.
  • the rule can be applied prior to the formation of the efficient frontier by, for example, applying the rule during data pre-selection.
  • heuristic rules can be applied that are not necessarily related to the biology in question. For example, one can apply the rule that only a given percentage of the portfolio can be represented by a particular gene or genes.
  • Commercially available software such as the Wagner Software readily accommodates these types of heuristics. This can be useful, for example, when factors other than accuracy and precision (e.g., anticipated licensing fees) have an impact on the desirability of including one or more genes.
  • One method of the invention involves comparing gene expression profiles for various genes (or portfolios) to ascribe prognoses.
  • the gene expression profiles of each of the genes comprising the portfolio are fixed in a medium such as a computer readable medium.
  • a medium such as a computer readable medium.
  • This can take a number of forms. For example, a table can be established into which the range of signals (e.g., intensity measurements) indicative of disease is input. Actual patient data can then be compared to the values in the table to determine whether the patient samples are normal or diseased.
  • patterns of the expression signals e.g., flourescent intensity
  • the gene expression patterns from the gene portfolios used in conjunction with patient samples are then compared to the expression patterns.
  • Pattern comparison software can then be used to determine whether the patient samples have a pattern indicative of recurrence of the disease. Of course, these comparisons can also be used to determine whether the patient is not likely to experience disease recurrence.
  • the expression profiles of the samples are then compared to the portfolio of a control cell. If the sample expression patterns are consistent with the expression pattern for recurrence of a colorectal cancer then (in the absence of countervailing medical considerations) the patient is treated as one would treat a relapse patient. If the sample expression patterns are consistent with the expression pattern from the normal/control cell then the patient is diagnosed negative for colorectal cancer.
  • the gene expression profiles of this invention can also be used in conjunction with other non-genetic diagnostic methods useful in cancer diagnosis, prognosis, or treatment monitoring.
  • diagnostic power of the gene expression based methods described above with data from conventional markers such as serum protein markers (e.g., carcinoembryonic antigen).
  • serum protein markers e.g., carcinoembryonic antigen
  • blood is periodically taken from a treated patient and then subjected to an enzyme immunoassay for one of the serum markers described above. When the concentration of the marker suggests the return of tumors or failure of therapy, a sample source amenable to gene expression analysis is taken.
  • tissue samples may be taken from areas adjacent to the tissue from which a tumor was previously removed. This approach can be particularly useful when other testing produces ambiguous results.
  • Articles of this invention include representations of the gene expression profiles useful for treating, diagnosing, prognosticating, and otherwise assessing diseases. These profile representations are reduced to a medium that can be automatically read by a machine such as computer readable media (magnetic, optical, and the like).
  • the articles can also include instructions for assessing the gene expression profiles in such media.
  • the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above.
  • the articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format. A graphical recordation is one such format. Clustering algorithms such as those incorporated in "DISCOVERY” and "INFER” software from Partek, Inc. mentioned above can best assist in the visualization of such data.
  • articles of manufacture are media or formatted assays used to reveal gene expression profiles. These can comprise, for example, microarrays in which sequence complements or probes are affixed to a matrix to which the sequences indicative of the genes of interest combine creating a readable determinant of their presence.
  • articles according to the invention can be fashioned into reagent kits for conducting hybridization, amplification, and signal generation indicative of the level of expression of the genes of interest for detecting colorectal cancer.
  • Kits made according to the invention include formatted assays for determining the gene expression profiles. These can include all or some of the materials needed to conduct the assays such as reagents and instructions.
  • Genes analyzed according to this invention are typically related to full-length nucleic acid sequences that code for the production of a protein or peptide.
  • identification of full-length sequences is not necessary from an analytical point of view. That is, portions of the sequences or ESTs can be selected according to well-known principles for which probes can be designed to assess gene expsession for the corresponding gene.
  • Example 1- Sample Handling and LCM.
  • Fresh frozen tissue samples were collected from patients who had surgery for colorectal tumors.
  • the samples that were used were from 63 patients staged with Duke's B according to standard clinical diagnostics and pathology. Clinical outcome of the patients was known. Thirty-six of the patients have remained disease-free for more than 3 years while 27 patients had tumor relapse within 3 years.
  • the tissues were snap frozen in liquid nitrogen within 20-30 minutes of harvesting, and stored at -80C° thereafter.
  • the samples were cut (6 ⁇ m), and one section was mounted on a glass slide, and the second on film (P.A.L.M.), which had been fixed onto a glass slide (Micro Slides Colorfrost, VWR Scientific, Media, PA).
  • the section mounted on a glass slide was after fixed in cold acetone, and stained with Mayer's Haematoxylin (Sigma, St. Louis, MO).
  • a pathologist analyzed the samples for diagnosis and grade. The clinical stage was estimated from the accompanying surgical pathology and clinical reports to verify the Dukes classification.
  • the section mounted on film was after fixed for five minutes in 100% ethanol, counter stained for 1 minute in eosin/100% ethanol (100 ⁇ g of Eosin in 100ml of dehydrated ethanol), quickly soaked once in 100% ethanol to remove the free stain, and air dried for 10 minutes.
  • the membrane LPC-MEMBRANE PEN FOIL 1.35 ⁇ m No 8100, P.A.L.M. GmbH Mikrolaser Technologie, Bernried, Germany
  • the slides were washed in DEP H 2 O, and the film was washed in RNase AWAY (Molecular Bioproducts, Inc., San Diego, CA) and rinsed in DEP H 2 O.
  • the slides were baked at +120°C for 8 hours, treated with TI-SAD (Diagnostic Products Corporation, Los Angeles, CA, 1:50 in DEP H 2 O, filtered through cotton wool), and incubated at +37°C for 30 minutes.
  • TI-SAD Diagnostic Products Corporation, Los Angeles, CA, 1:50 in DEP H 2 O, filtered through cotton wool
  • the tissue sections mounted on film were used for LCM. Approximately 2000 epithelial cells/sample were captured using the PALM Robot-Microbeam technology (P.A.L.M. Mikrolaser Technologie, Carl Zeiss, Inc., Thornwood, NY), coupled into Zeiss Axiovert 135 microscope (Carl Zeiss Jena GmbH, Jena, Germany). The surrounding stroma in the normal mucosa, and the occasional intervening stromal components in cancer samples, were included. The captured cells were put in tubes in 100% ethanol and preserved at -80°C.
  • Zymo-Spin Column (Zymo Research, Orange, CA 92867) was used to extract total RNA from the LCM captured samples. About 2 ng of total RNA was resuspended in 10 ul of water and 2 rounds of the T7 RNA polymerase based amplification were performed to yield about 50 ug of amplified RNA.
  • Example 3- cDNA Microarray Hybridization and Quantitation.
  • a set of cDNA microarrays consisting of approximately 23,000 human cDNA clones was used to test the samples by use of the humanU133a chip obtained and commercially available from Affymetrix, Inc. Total RNA obtained and prepared as outlined above and applied to the chips and analyzed by Agilent BioAnalyzer according to the manufacturer's protocol. All 63 samples passed the quality control standards and the data were used for marker selection.
  • Chip intensity data was analyzed using MAS Version 5.0 software commercially available from Affymetrix, Inc. ("MAS 5.0''). An unsupervised analysis was used to identify two genes that distinguish patients that would relapse from those who would not as follows.
  • the chip intensity data obtained as described was the input for the unsupervised clustering software commercially available as PARTEK version 5.1 software.
  • This unsupervised clustering algorithm identified a group of 20 patients with a high frequency of relapse (13 relapsers and 7 survivors). From the original 23,000 genes, t-testing analysis selected 276 genes that significantly differentially expressed in these patients. From this group, two genes were selected that best distinguish relapsing patients from those that do not relapse: Human intestinal peptide-associated transporter (Seq. ID. No.
  • MHC II-DR-B was chosen. These genes are down-regulated in relapsers. MHC II-DR-B (Seq. ID No. 2) also had the smallest p-value ( Figure 3a).

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054508A2 (fr) * 2003-12-01 2005-06-16 Ipsogen Profilage de l'expression des genes dans le cancer du colon par microreseaux d'adn et correlation avec des parametres de survie et histocliniques
WO2008045133A2 (fr) * 2006-03-03 2008-04-17 Veridex, Llc Analyse moléculaire pour prévoir une récidive du cancer du côlon de stade dukes b

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003001985A2 (fr) * 2001-06-28 2003-01-09 Dermtech International Methode de depistage de melanome
US20040191782A1 (en) * 2003-03-31 2004-09-30 Yixin Wang Colorectal cancer prognostics
CA2475769C (fr) * 2003-08-28 2018-12-11 Veridex, Llc Pronostic du cancer colo-rectal
US7183057B2 (en) * 2004-03-31 2007-02-27 Dermtech International Tape stripping methods for analysis of skin disease and pathological skin state
DE102005026710A1 (de) * 2005-06-09 2006-12-14 Basf Ag Verfahren zum Testen von Substanzen oder Substanzgemischen, dessen Verwendung und entsprechende Analysekits
NZ544432A (en) * 2005-12-23 2009-07-31 Pacific Edge Biotechnology Ltd Prognosis prediction for colorectal cancer using a prognositc signature comprising markers ME2 and FAS
WO2008021183A2 (fr) * 2006-08-10 2008-02-21 Millennium Pharmaceuticals, Inc. Procédés d'identification, d'évaluation, et de traitement de patients soumis à une thérapie anticancéreuse
NZ580710A (en) * 2007-05-04 2012-06-29 Dermtech Int Diagnosis of melanoma by nucleic acid analysis
FR2919060B1 (fr) * 2007-07-19 2012-11-30 Biomerieux Sa Procede de dosage de l'ezrine pour le diagnostic in vitro du cancer colorectal.
FR2919065B1 (fr) * 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de l'apolipoproteine ai pour le diagnostic in vitro du cancer colorectal
FR2919064B1 (fr) * 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de l'apolipoproteine all pour le diagnostic in vitro du cancer colorectal
FR2919062B1 (fr) * 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de l'aminoacylase 1 pour le diagnostic in vitro du cancer colorectal.
FR2919061B1 (fr) * 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage de la plastine-i pour le diagnostic in vitro du cancer colorectal.
FR2919063B1 (fr) * 2007-07-19 2009-10-02 Biomerieux Sa Procede de dosage du leucocyte elastase inhibitor pour le diagnostic in vitro du cancer colorectal.
WO2009019368A2 (fr) * 2007-07-19 2009-02-12 bioMérieux Procede de dosage de la liver fatty acid-binding protein, de l'ace et du ca19-9 pour le diagnostic in vitro du cancer colorectal
ES2332557B1 (es) * 2007-12-04 2011-02-02 Universidad Autonoma De Madrid Huella genomica para el pronostico de la evolucion de adenocarcinoma colorectal.
US20090192045A1 (en) * 2008-01-22 2009-07-30 Yuqiu Jiang Molecular staging of stage ii and iii colon cancer and prognosis
CN102089444A (zh) 2008-05-14 2011-06-08 德玛泰克国际公司 利用核酸分析方法来诊断黑素瘤和太阳能雀斑
FR2933773B1 (fr) * 2008-07-10 2013-02-15 Biomerieux Sa Procede de dosage de la proteine disulfide isomerase pour le diagnostic in vitro du cancer colorectal
WO2010025341A2 (fr) * 2008-08-28 2010-03-04 Dermtech International Détermination de tranches d’âge d’échantillons cutanés
CN102766679B (zh) * 2011-05-04 2015-02-25 上海人类基因组研究中心 通过二基因表达情况预测大肠癌术后无复发生存的检测方法、探针组和诊断试剂盒
US11976332B2 (en) 2018-02-14 2024-05-07 Dermtech, Inc. Gene classifiers and uses thereof in non-melanoma skin cancers
EP3948290A4 (fr) 2019-03-26 2023-08-09 Dermtech, Inc. Nouveaux classificateurs de gènes et leurs utilisations pour des cancers de la peau

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000019206A1 (fr) * 1998-10-01 2000-04-06 The Regents Of The University Of California Ykl-40 en tant q ue marqueur et indicateur de pronostic pour le cancer
WO2001094629A2 (fr) * 2000-06-05 2001-12-13 Avalon Pharmaceuticals Determination de gene du cancer et recherche therapeutique utilisant des ensembles de genes signature
EP1349104A2 (fr) * 2002-03-29 2003-10-01 Ortho-Clinical Diagnostics, Inc. Méthode pour la sélection d'un ensemble des marqueurs pour une application diagnostique
EP1355150A2 (fr) * 2002-03-29 2003-10-22 Ortho-Clinical Diagnostics, Inc. Panel d'acide nucléique pour le diagnostic du cancer
US20040191782A1 (en) * 2003-03-31 2004-09-30 Yixin Wang Colorectal cancer prognostics

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8822228D0 (en) * 1988-09-21 1988-10-26 Southern E M Support-bound oligonucleotides
US5242974A (en) * 1991-11-22 1993-09-07 Affymax Technologies N.V. Polymer reversal on solid surfaces
US5527681A (en) * 1989-06-07 1996-06-18 Affymax Technologies N.V. Immobilized molecular synthesis of systematically substituted compounds
US5143854A (en) * 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US5424186A (en) * 1989-06-07 1995-06-13 Affymax Technologies N.V. Very large scale immobilized polymer synthesis
IL103674A0 (en) * 1991-11-19 1993-04-04 Houston Advanced Res Center Method and apparatus for molecule detection
US5412087A (en) * 1992-04-24 1995-05-02 Affymax Technologies N.V. Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces
US5384261A (en) * 1991-11-22 1995-01-24 Affymax Technologies N.V. Very large scale immobilized polymer synthesis using mechanically directed flow paths
US5554501A (en) * 1992-10-29 1996-09-10 Beckman Instruments, Inc. Biopolymer synthesis using surface activated biaxially oriented polypropylene
US5472672A (en) * 1993-10-22 1995-12-05 The Board Of Trustees Of The Leland Stanford Junior University Apparatus and method for polymer synthesis using arrays
US5429807A (en) * 1993-10-28 1995-07-04 Beckman Instruments, Inc. Method and apparatus for creating biopolymer arrays on a solid support surface
US5545531A (en) * 1995-06-07 1996-08-13 Affymax Technologies N.V. Methods for making a device for concurrently processing multiple biological chip assays
US6812339B1 (en) 2000-09-08 2004-11-02 Applera Corporation Polymorphisms in known genes associated with human disease, methods of detection and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000019206A1 (fr) * 1998-10-01 2000-04-06 The Regents Of The University Of California Ykl-40 en tant q ue marqueur et indicateur de pronostic pour le cancer
WO2001094629A2 (fr) * 2000-06-05 2001-12-13 Avalon Pharmaceuticals Determination de gene du cancer et recherche therapeutique utilisant des ensembles de genes signature
EP1349104A2 (fr) * 2002-03-29 2003-10-01 Ortho-Clinical Diagnostics, Inc. Méthode pour la sélection d'un ensemble des marqueurs pour une application diagnostique
EP1355150A2 (fr) * 2002-03-29 2003-10-22 Ortho-Clinical Diagnostics, Inc. Panel d'acide nucléique pour le diagnostic du cancer
US20040191782A1 (en) * 2003-03-31 2004-09-30 Yixin Wang Colorectal cancer prognostics

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HEGDE ET AL: "Identification of tumor markers in models of human colorectal cancer using a 19200-element complementary DNA microarray" CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, MD, US, vol. 61, no. 21, 1 November 2001 (2001-11-01), pages 7792-7797, XP002210615 ISSN: 0008-5472 *
ZHANG L ET AL: "Gene expression profiles in normal and cancer cells" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 276, 23 May 1997 (1997-05-23), pages 1268-1272, XP002083785 ISSN: 0036-8075 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054508A2 (fr) * 2003-12-01 2005-06-16 Ipsogen Profilage de l'expression des genes dans le cancer du colon par microreseaux d'adn et correlation avec des parametres de survie et histocliniques
WO2005054508A3 (fr) * 2003-12-01 2006-05-18 Ipsogen Profilage de l'expression des genes dans le cancer du colon par microreseaux d'adn et correlation avec des parametres de survie et histocliniques
WO2008045133A2 (fr) * 2006-03-03 2008-04-17 Veridex, Llc Analyse moléculaire pour prévoir une récidive du cancer du côlon de stade dukes b
WO2008045133A3 (fr) * 2006-03-03 2008-09-12 Veridex Llc Analyse moléculaire pour prévoir une récidive du cancer du côlon de stade dukes b

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AR043808A1 (es) 2005-08-17
CA2464894C (fr) 2014-02-11
KR20040088384A (ko) 2004-10-16
JP2004329211A (ja) 2004-11-25
BRPI0403169A (pt) 2005-09-20
EP1526186B1 (fr) 2008-10-22
CA2464894A1 (fr) 2004-09-30
KR101088583B1 (ko) 2011-12-05
ES2316932T3 (es) 2009-04-16
MXPA04003044A (es) 2005-07-01
JP4913331B2 (ja) 2012-04-11
CN100393886C (zh) 2008-06-11
AU2004201348A1 (en) 2004-10-14
ATE412070T1 (de) 2008-11-15
CL2004000700A1 (es) 2005-02-25
AU2004201348B2 (en) 2008-04-10
CN1542143A (zh) 2004-11-03
DE602004017265D1 (de) 2008-12-04
US20040191782A1 (en) 2004-09-30

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